egfp open reading frame orf  (Addgene inc)

Journal: bioRxiv

Article Title: A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release

doi: 10.1101/2024.05.24.595612

Figure Lengend Snippet: a, Schematic of the fluorescent reporter construct for transcriptional activation; pInducer20-eGFP. An eGFP open reading frame is placed after a Tet Responsive Element (TRE) sequence. Transcription of eGFP can either be facilitated by activation of the co-expressed reverse tet-transactivator rTA3 by addition of doxycycline (1), or by introduction of transcriptional activator dCas9-VPR with a sgRNA targeting the TRE sequence (2). b,c Fluorescence microscopy images (b) and flow cytometry analysis (c) of HEK293T cells expressing the pInducer20-eGFP reporter construct, 48 hours after addition of doxycycline (0.5 µg/ml), or transfection with plasmids encoding for dCas9-VPR with non-targeting (NT) sgRNAs, targeting (T) sgRNAs, or targeting MS2-sgRNAs. Doxycyline and dCas9-VPR with targeting sgRNAs increase eGFP expression. MFI: mean fluorescence intensity. Scalebar represents 200 μm. Means + SD, n = 3, One-way ANOVA with post-hoc Dunnett’s multiple comparison test. d, Flow cytometry analysis of HEK293T cells expressing the pInducer20-eGFP reporter construct, 48 hours after addition of EVs from HEK293T expressing dCas9-VPR alongside either MCP-PhoCl-CD63 or MCP-PhoCl-CD9, in combination with non-targeting- (NT), or targeting (T) sgRNAs. Both loading constructs facilitate EV-mediated functional dCas9- VPR delivery, resulting in a significant increase in eGFP mean fluorescence intensity (MFI). 4.0x10 11 EVs per well. Means + SD, n = 5, One-way ANOVA with post-hoc Dunnett’s multiple comparison test. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: For the generation of a lentiviral doxycycline-inducible eGFP expression construct, an eGFP open reading frame was transferred from pDONR221- eGFP (Addgene #25899) into pInducer20 (Addgene #44012) using the Gateway LR Clonase II Enzyme Mix (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Techniques: Construct, Activation Assay, Sequencing, Fluorescence, Microscopy, Flow Cytometry, Expressing, Transfection, Comparison, Functional Assay

Journal: eLife

Article Title: Effect of SARS-CoV-2 proteins on vascular permeability

doi: 10.7554/eLife.69314

Figure Lengend Snippet: (a) Bright-field and fluorescent image of infected eGFP HUVEC, scale bar: 50 µm; ( b ) changes in barrier functions as a result of SARS-CoV-2 proteins were assessed by trans-epithelial-endothelial electrical resistance (TEER) measurement. Note the statistical differences compared to the untreated control condition, assessed by F-statistic with two-way ANOVA test, followed by the Holm–Sidak test for multiple comparisons; ( c ) color map showing a gradual decrease in TEER values compared to the untreated condition at day 3; ( d ) immunocytochemistry (ICC) for CD31 (green) and Hoechst (blue) for the three specified conditions, scale bar: 20 µm; ( e ) analysis of CD31 expression levels.

Article Snippet: Plasmids encoding the SARS-CoV-2 open reading frames proteins and eGFP control were a kind gift of Nevan Krogan (Addgene plasmid #141367–141395).

Techniques: Infection, Control, Immunocytochemistry, Expressing

Journal: eLife

Article Title: Effect of SARS-CoV-2 proteins on vascular permeability

doi: 10.7554/eLife.69314

Figure Lengend Snippet: (a) Confocal reconstructions of HUVEC stained for von Willebrand factor (VWF) (green) and Hoechst (blue) for three conditions: control (untreated), eGFP, and nsp5_c145a, scale bar: 20 µm; ( b ) analysis of VWF expression levels; ( c ) fold change of interleukin (IL)-6 in response to the different proteins.

Article Snippet: Plasmids encoding the SARS-CoV-2 open reading frames proteins and eGFP control were a kind gift of Nevan Krogan (Addgene plasmid #141367–141395).

Techniques: Staining, Control, Expressing

Journal: bioRxiv

Article Title: Inducible Yeast Two-Hybrid with Quantitative Measures

doi: 10.1101/2021.07.01.450807

Figure Lengend Snippet: (A) Map of the 7-kb modified pGBKT7 vector with CUP1 promoter (copper arrow). Modified CUP1 promoter sequence to remove restriction sites is shown below. (B) The CUP1 promoter increased expression in response to an increase in CuSO 4 concentration. Western blot data showed a change in protein expression as CuSO 4 concentrations increased from 0 (uninduced) to 50, 100, and 200 μM (induced). Normalized expression levels were plotted relative to 0mM CuSO 4 (uninduced) and a best fit line was overlayed. (C) Western blot data showed greater expression when using an induced CUP1 promoter compared to the ADH1 promoter, and greater expression of NXF1-B than NXF1-C. Normalized expression levels were plotted as raw values and a best fit line was plotted only for CUP1 driven NXF1-B and NXF1-C (NXF1-B, NXF1 B 6 ; NXF1-C, NXF1 CAST ).

Article Snippet: Plasmids have been deposited with Addgene, including pGBK- CUP1 empty vector (Addgene 169710) and pGBK- CUP1 with open reading frames for EGFP (170210), mCherry (170264), mGrape3 (170265), Nxf1-B (170266), and Nxf1-C (170267).

Techniques: Modification, Plasmid Preparation, Sequencing, Expressing, Concentration Assay, Western Blot